1. Field of the Invention
The present invention relates to a method for immobilizing a compound on magnetic beads.
2. Description of the Related Art
Magnetic beads are extremely useful for obtaining a product of interest, such as a protein, a DNA, or a cell, from a mixture of various substances. For example, when a specific protein is obtained from a cell extract, a ligand that specifically binds to a protein of interest is immobilized on the surface of each of magnetic beads. The thus ligand-immobilized magnetic beads are then added to the extract, and the protein of interest is allowed to bind to the magnetic beads via the ligand. Thereafter, the magnetic beads are recovered utilizing magnetic force and the like, thereby obtaining the protein of interest. Hence, if taking advantage of the characteristics of magnetic beads, a substance of interest can be efficiently obtained from a mixture comprising various substances.
Among biological substances, in a case in which an antibody having a desired specificity is selected from an antibody library, the use of magnetic beads is particularly effective. For example, in a case in which a protein is used as an antigen and in which an antibody specifically binding to such antigen is selected from a library, if magnetic beads on the surface of each of which a tosyl group or an epoxy group is present are used, covalent bonds can be formed between such functional groups and many free amino groups existing in the protein. Thus, the antigenic protein can be easily immobilized on the magnetic beads. However, in the case of a compound having almost no amino groups (for example, a low-molecular-weight compound such as a lipid), since it is difficult to bind the compound to the magnetic beads mediated by such amino groups, other methods should have been applied. Examples of such other methods attempted to replace the above-mentioned method include: a method mediated by a carrier protein; a method utilizing a biotin-avidin bond involving biotinylation; a method using magnetic beads having hydrophobic functional groups; a method of artificially introducing an amino group into an antigen and a method using a photoreactive crosslinking reagent (US2006194251). However, the use of the method of binding a carrier protein to an antigen, the method of biotinylating an antigen, or the like, has been problematic in that these methods may affect the steric structure of an antigenic molecule, or in that an antibody may be obtained selectively with respect to such biotinylated portion or carrier protein. Moreover, if the method using magnetic beads having hydrophobic functional groups is applied to the selection of an antibody from an antibody library constituted with living cells (see, for example, an antibody library prepared by an ADLib method, US20060183225; Seo et al, Nature Biotech. 23: 731-735, 2005), antigens are easily released from the beads, and the thus released antigens have lethal effects on the cells constituting the library. Furthermore, since the method of artificially introducing amino groups into antigens and then directly binding the antigens to magnetic beads has required a chemical synthesis comprising complicated steps, this method has required enormous time and expense.
In contrast to the above-described methods, a method of allowing antigens to bind to magnetic beads using a photoreactive crosslinking reagent (a photoreactive compound) has been expected to be an effective, versatile means for immobilizing various types of compounds on magnetic beads. However, as a matter of fact, the efficiency of immobilizing antigens on magnetic beads has been significantly low, under conditions for binding the antigens to the conventionally used solid phase or agarose beads (for example, under ultraviolet irradiation and/or crosslinking conditions). As a result, the expected effects could not be obtained.
Under the above-mentioned circumstances, using magnetic beads, in order to select from an antibody library, an antibody reacting with an antigen which has been hardly immobilized on magnetic beads by the conventional method, it has been necessary to establish a modified method for stably and efficiently immobilizing a compound serving as an antigen on magnetic beads.